Genome-based analysis of the family Paracoccaceae and description of Ostreiculturibacter nitratireducens gen. nov., sp. nov., isolated from an oyster farm on a tidal flat

Two bacterial strains, designated FR2A1T and MT2-5-38, were isolated from the surface sediments of an oyster farm on a tidal flat in Quanzhou Bay, China. Both strains were Gram-stain-negative, rod-shaped, aerobic, catalase-positive, and oxidase-positive. The 16S rRNA gene sequences of the two strains were 100% identical and had the highest similarity (97.1%) with Phaeovulum vinaykumarii JA123T. The average nucleotide identity (ANI) value and digital DNA–DNA hybridization (DDH) value indicated that the two strains belonged to a single species. Gene annotation revealed that the two strains contained a gene cluster for nitrate reduction and a gene cluster for sulfur oxidation, indicating a possible role in N and S cycling in the tidal flat sediment. The phylogeny inferred from the 16S rRNA gene and 120 conserved proteins indicated that the two strains formed a distinct monophyletic clade within the family Paracoccaceae. The respiratory quinone was Q-10. The major fatty acids consisted of summed feature 8 (C18:1ω7c and/or C18:1ω6c) and C18:0. The polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, and several unidentified phospholipids. Based on the above characteristics, strains FR2A1T and MT2-5-38 represent a novel genus and a novel species, for which we propose the name Ostreiculturibacter nitratireducens gen. nov., sp. nov. The type strain is FR2A1T (=MCCC 1K08809T = KCTC 8317T). Phylogenomic analysis of 1,606 high-quality genomes of the family Paracoccaceae, including type strains, non-type strains, and uncultivated bacteria, was performed using the Genome Taxonomic Database Toolkit (GTDB-Tk), and the average amino acid identity (AAI) value of the phylogenetic clade was estimated. We found that 35 species of the family Paracoccaceae needed re-classification, and an AAI value of 70% was chosen as the genus boundary within the family Paracoccaceae.


Introduction
The family Paracoccaceae (illegitimate synonym: "Rhodobacteraceae") comprises the majority of the Alphaproteobacteria in marine habitats and displays a large phenotypic, genotypic, and metabolic diversity (Simon et al., 2017).In previous studies, the assignment of isolates (or genomes) to a species and/or genus of the family Paracoccaceae mainly depended on 16S rRNA gene phylogeny, which often resulted in misclassifications (Liang et al., 2021).To give a better resolution of the taxonomy of the family Paracoccaceae, phylogenomic analysis based on bacterial ubiquitous gene sets should be performed (Hördt et al., 2020;Liang et al., 2021;Zhang et al., 2023).Thus, the family Roseobacteraceae was split off from the representatives of the family Rhodobacteraceae in 2021, based on core-genome phylogeny (Liang et al., 2021).
In 2022, the name Paracoccaceae was proposed to replace the name Rhodobacteraceae, which is illegitimate because it contravenes Rule 51 of the International Code of Nomenclature of Prokaryotes (Göker, 2022).At the time of writing, 92 genera were included in the family Paracoccaceae.1 Bacterial species delineation based on genomic metrics is generally accepted, such as average nucleotide identity (ANI) and digital DNA-DNA hybridization (DDH) estimates, whereas a consensus genomic metric boundary for a genus delineation of the family Paracoccaceae is still lacking.In addition, misclassifications have occurred when using 16S rRNA gene phylogeny or including a part of type strains in the phylogenomic reconstruction.Thus, a comprehensive phylogenomic analysis of the family Paracoccaceae is necessary, using large datasets.
In this study, two strains, designated MT2-5-38 and FR2A1 T , were isolated from the surface sediments of an oyster farm on a tidal flat in Quanzhou Bay, Fujian Province, China, in 2019 and 2023, respectively.The 16S rRNA gene sequence of the two strains was found to be 100% identical, suggesting that the organism may represent a novel species affiliated to the family Paracoccaceae.This study aimed to determine the taxonomic position of the strains.In addition, a comprehensive phylogenomic analysis of the family Paracoccaceae was performed based on the available genomes, including type strains, non-type strains, and uncultivated bacteria.The combination of phylogenomic analysis and average amino acid identity (AAI) metrics was used to elucidate the taxonomy of the family Paracoccaceae.

Strain isolation and cultivation
Two bacterial strains designated MT2-5-38 and FR2A1 T were isolated from surface sediments of an oyster farm on a tidal flat in Quanzhou Bay (24°86′ N, 118°68′ E), Fujian Province, China, in March 2019 (Huang et al., 2021) and in August 2023, respectively.For the isolation of strain FR2A1 T , 1 g surface sediment was diluted in 9 mL sterile seawater, and samples of 10× serial dilutions were spread onto marine R2A agar culture medium (R2A powder dissolved with natural seawater, adding 1.5% agar [BD]) and incubated at 28°C for 14 days.Strain FR2A1 T was picked and streaked onto MA (Marine Broth 2216 [BD] plus 1.5% Agar [BD]).Strain MT2-5-38 was isolated using a similar protocol (Huang et al., 2021).Briefly, 0.1 g surface sediment was diluted in 0.9 mL sterile seawater, and dilutions were spread onto MA plates.
The strains were stored at −80°C with 20% glycerol (v/v) and deposited in the Marine Culture Collection of China (MCCC) and the Korean Collection for Type Cultures (KCTC).

Phylogeny of the 16S rRNA gene
The genomic DNA of strain FR2A1 T was extracted from fresh cells using the Bacterial Genomic Extraction Kit (SaiBaisheng, Co., Ltd., Shanghai, China).The 16S rRNA gene was amplified using bacterial primers Eubac27F and 1492R (Delong, 1992) with Ex Taq (TaKaRa) in 50 μL PCR system.Then, the PCR product was ligated into the pMD19-T vector (TaKaRa) and chemically transformed into competent Escherichia coli DH5α cells.A positive clone was selected and used for Sanger sequencing with the vector primer.The nearly complete 16S rRNA gene sequence of strain FR2A1 T was assembled using DNAMAN version 8.The partial 16S rRNA gene sequence of strain MT2-5-38 was determined in our previous study and deposited in GenBank under accession number MT829653 (Huang et al., 2021).
The close relatives of strain FR2A1 T and strain MT2-5-38 were searched, and their 16S rRNA gene sequences were downloaded from the EzBioCloud database (Yoon et al., 2017a) and the NCBI nucleotide database. 2Then, the sequences were subjected to multiple alignments by the Clustal W program implemented in MEGA 7.0 (Kumar et al., 2016).The phylogenetic tree was constructed based on two algorithms, neighbor-joining and maximum-likelihood, with 1,000 bootstraps using MEGA 7.0.The models used in neighbor-joining tree and maximum-likelihood tree were maximum composite likelihood (MCL) and K2 + G + I, respectively.

Whole genome sequencing and genome annotation
The whole genome sequences of strain FR2A1 T and strain MT2-5-38 were determined using the Illumina NovaSeq platform (Shanghai Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China).The raw paired-end reads were trimmed using sickle3 with a length of 50 bp (−l 50) and quality score of 20 (−q 20).The clean reads were then assembled into contigs using SPAdes v3.8.0 (Bankevich et al., 2012).Contigs shorter than 1 kb were removed from the assembly.The complete 16S rRNA gene sequence was extracted from the whole genome sequences using RNAmmer (Lagesen et al., 2007).Genome quality (completeness and contamination) and classification were evaluated using CheckM v1.2.0 (Parks et al., 2015).
Gene prediction was performed using GeneMarkS (Besemer et al., 2001), and gene annotation was carried out using the RAST server (Aziz et al., 2008) and KAAS system. 4 Functional proteins with the best similarities to close relatives were searched using the BLASTp program against the nr database with e-value cutoff of 1e−5 (Camacho et al., 2009).
The genomes affiliated to the family Paracoccaceae were searched and downloaded from the genome portal of NCBI7 as of 25 August 2023.A total of 4,316 genomes, including type strains, non-type strains, and uncultivated bacteria, were obtained and used for genome quality estimation.The genome quality was estimated using CheckM v.1.2.0 (Parks et al., 2015).Genomes with <90% completeness and >5% contamination were excluded from the following study.The phylogenomic tree was constructed using the GTDB-Tk 1.3.0 based on 120 ubiquitously conserved bacterial proteins (Chaumeil et al., 2019).The tree was visualized using the Interactive Tree of Life (iTOL) online (Letunic and Bork, 2007).

Phenotypic characterization
Strains FR2A1 T and MT2-5-38, together with the reference strain Phaeovulum vinaykumarii JA123 T (=DSM 18714 T ), were maintained under identical conditions for phenotypic comparison.Colony morphology was recorded on MA after incubation at 30°C for 2 days.Gram staining was carried out using a Gram staining kit (Hangzhou Microbial Reagent, Co. Ltd.).Catalase activity was tested using a 3% H 2 O 2 solution.Oxidase activity was tested using the oxidase reagent (bioMérieux, France).The growth temperature range was determined under various temperatures (4, 10, 15, 20, 25, 28, 30, 35, 40, and 45°C) for 1 week.Anaerobic culture was tested in 10 mL MB in 50 mL anaerobic flasks according to our previously documented method (Liu et al., 2019).Physiological and biochemical characterization was carried out at 30°C using API ZYM, API 20NE, and API 20E strips according to the manufacturer's instructions (bioMérieux, France).

Chemotaxonomic characterization
The respiratory quinone of strain FR2A1 T was extracted as described previously (Komagata and Suzuki, 1987) and assayed by using reversed-phase high-performance liquid chromatography (Agilent 1200).For cellular fatty acid analyses, strains FR2A1 T , MT2-5-38, and P. vinaykumarii JA123 T were cultured in MB at 30°C with shaking at 160 rpm for 2 days.The biomass was collected using centrifugation at 6,000 rpm for 10 min.The cellular fatty acids were saponified, methylated, extracted, and identified following the standard MIDI protocol (Sherlock Microbial Identification System, version 6B).For polar lipid analysis, strain FR2A1 T was cultured in 100 mL MB medium for 2 days, and cells were harvested by centrifugation at 6,000 rpm.Polar lipids were extracted using a chloroform/methanol system and analyzed using one-and two-dimensional TLC using Merck silica gel 60 F254 aluminumbacked thin-layer plates.Phospholipids were detected by spraying the plate with molybdenum blue.

Nucleotide sequences
The GenBank/EMBL/DDBJ accession numbers of the 16S rRNA gene sequence of strains FR2A1 T and MT2-5-38 are OR533672 and MT829653, respectively.The whole genome sequences of strain FR2A1 T and strain MT2-5-38 have been deposited at GenBank under the accession numbers JAVQHL000000000 and JAVQHM000000000, respectively.

16S rRNA gene sequence phylogeny
The nearly complete (1,391 bp) 16S rRNA gene sequence of strain FR2A1 T was obtained using Sanger sequencing.It had 100% identical sequence similarity with that of strain MT2-5-38.A sequence similarity search showed that strain FR2A1 T had the highest 16S rRNA gene similarity (97.1%) with Phaeovulum vinaykumarii JA123 T .Phylogenetic analysis based on the 16S rRNA gene indicated that strain FR2A1 T and strain MT2-5-38 formed a monophyletic clade distinct from closely related genera affiliated to the family Paracoccaceae and may be considered a new species within a new genus (Figure 1; Supplementary Figure S1).Based on the 16S rRNA gene sequence similarity, P. vinaykumarii JA123 T (=DSM 18714 T ) was chosen as a reference strain.

BOX-PCR fingerprinting
The BOX-PCR fingerprinting profile of strain FR2A1 T and strain MT2-5-38 showed distinctive electrophoresis patterns (Supplementary Figure S2), confirming that they are not clonal.

Genomic characteristics
The whole genome sequences of strains FR2A1 T and MT2-5-38 were determined.The genome size of strain FR2A1 T was 4,009,665 bp on 12 contigs (>1 kb).The genome size of strain MT2-5-38 was 3,901,132 bp on 14 contigs (>1 kb; Table 1).The genomic G + C content of strain FR2A1 T and strain MT2-5-38 was 65.7 and 65.6%, respectively.The ANI value and DDH estimate between strains FR2A1 T and MT2-5-38 were estimated to be 99.5 and 96.7%, respectively, which strongly supports that they belong to the same species.The ANI value and DDH estimate between strain FR2A1 T and the closest reference strain, P. vinaykumarii JA123 T , were 74.5 and 21.0%, respectively.
Functional gene prediction showed 3,919 and 3,803 genes in strain FR2A1 T and strain MT2-5-38, respectively.Both strains contained a full set of genes for respiratory nitrate reduction (narI, narJ, narH, and narG), responsible for the reduction of nitrate to Phylogenetic analysis based on 16S rRNA gene sequences.The tree was constructed using the neighbor-joining method.Rhodospirillum rubrum ATCC 11170 T (CP000230) was selected as the outgroup.The new isolates are marked in bold.Bootstrapping was carried out with 1,000 replicates.Branch node values below 70% are not shown.Bar, 0.02 represented the nucleotide substitution per position.

Phylogenomic analysis of the family Paracoccaceae
A phylogenomic tree including 1,606 high-quality genomes was constructed based on 120 conserved bacterial proteins using GTDB-Tk 1.3.0,and the AAI values of the phylogenetic clades were estimated.The studied genomes included not only the representatives of the family Paracoccaceae but also the representatives of the family Roseobacteraceae, indicating that taxonomic correction was needed.The family Roseobacteraceae was proposed in 2021 based on core-genome phylogeny; it was split off from the family Paracoccaceae (Liang et al., 2021).The genomic characteristics (genome size, G + C content, and AAI values) are listed in Supplementary Table S2.The two families formed multiple clades (Figure 2; Supplementary Figures S5, S6).Our study did not resolve the two families.Whether the family Roseobacteraceae needs to be re-merged into the Paracoccaceae needs further investigation.
The phylogenomic tree constructed based on the 120 ubiquitously conserved bacterial proteins showed that strain FR2A1 T formed a distinct monophyletic branch with an uncultivated bacterium bin.37 (GCA_024742695.1),a bacterium found in the phycosphere of a toxic marine dinoflagellate (Alexandrium tamarense), which was separated from other genera affiliated to the family Paracoccaceae (Supplementary Figure S5).The genome size of the bacterium bin.37 was 4.2 Mb with a genomic G + C content of 69.2%.Gene prediction showed 4,125 functional genes in the bacterium bin.37.A gene cluster for sulfur oxidation (SoxABCDYZ) was also annotated, but the gene cluster for nitrate reduction was not found (Supplementary Table S1).The ANI and AAI values between strain FR2A1 T and bacterium bin.37 were 75.9 and 71.8%, respectively.Thus, our phylogenomic analysis and genomic relatedness strongly supported that the bacterium bin.37 belonged to the same genus as strain FR2A1 T .In the course of the phylogenomic analysis of the 1,606 genomes of the family Paracoccaceae, we found discrepancies in the taxonomic positions of a few members, as elucidated below.

Thermohalobaculum and Limibaculum
The genus Thermohalobaculum with type species Thermohalobaculum xanthum was proposed in 2021 (Pan et al., 2021).T. xanthum M0105 T and Limibaculum sediminis FT325 T are grouped together in the phylogenomic tree (Figure 2).The AAI and ANI values between T. xanthum M0105 T and L. sediminis FT325 T were 81.7 and 82.0%, respectively.The phylogeny of the 16S rRNA gene sequences placed the two strains into a highly supported clade (bootstrap of 99%), which is neighbored by Limibaculum halophilum (type species of Limibaculum) and Rubrimonas representatives (Supplementary Figure S7).The analysis supported that T. xanthum and L. sediminis could be merged into a single genus.Thus, we propose the transfer of L. sediminis to the genus Thermohalobaculum as Thermohalobaculum sediminis comb.nov.The genome size of Thermohalobaculum was 4.1-4.3Mbp.The genomic G + C content of Thermohalobaculum was 67.9-69.6%.

Pontivivens, Monaibacterium, and Pontibrevibacter
Pontivivens currently contains two species, P. insulae (type species) and P. ytuae (Parte et al., 2020).However, the two species did not form a node in the phylogenomic tree (Figure 2).Monaibacterium marinum C7 T and Pontibrevibacter nitratireducens h42 T formed a highly supported clade, sharing AAI and ANI values of 81.5 and 78.2%, respectively, showing that they could be considered representatives of the same genus.The four species, P. insulae, P. ytuae, M. marinum, and Pontibrevibacter nitratireducens, were tightly clustered with 100% bootstrap values, sharing AAI values of 70.0-100%.The AAI values between Rubricella aquisinus DSM 103377 T and the eight close relatives were 65.3-66.5%.Thus, it is reasonable to merge the four species P. insulae, P. ytuae, M. marinum, and Pontibrevibacter nitratireducens into a single genus, separated from the genus Rubricella.Based on priority, M. marinum and P. nitratireducens should be merged into the genus Pontivivens as Pontivivens marinum comb.nov.and Pontivivens nitratireducens comb.nov., respectively.The genome size of Pontivivens representatives was 3.1-4.2Mbp.The genomic G + C content was 58.9-67.2%(Supplementary Table S2).

Amylibacter and Neptunicoccus
The phylogeny of Amylibacter genomes indicated multi-phyletic clades (Figure 3A).The heatmap of AAI values supported six distinct groups, corresponding to the six phylogenetic clades in the tree (Figure 4).First, A. ulvae KCTC 32465 T and A. kogurei 4G11 T formed a highly supported clade, sharing an AAI value of 92.6%, which was Summed features are groups of two or three fatty acids that cannot be separated by GLC using the MIDI system.Summed feature 2 comprised C12:0 aldehyde and unknown 10.9283, summed feature 3 comprised C16:1ω7c and C16:1ω6c, and summed feature 8 comprised C18:1 ω7c and/or C18:1 ω6c.The major fatty acids (>10%) are maked bold.3A).

Halocynthiibacter and Paenihalocynthiibacter
Phylogenetic analysis placed Halocynthiibacter namhaensis RA2-3 T and Paenihalocynthiibacter styelae MYP1-1 T into a closely related clade, sharing an AAI value of 81%, indicating that the strains belonged to the same genus (Figure 3B).Cochlodiniinecator piscidefendens M26A2M T shared AAI values of 68.8 and 68.7% with Halocynthiibacter namhaensis RA2-3 T and Paenihalocynthiibacter styelae MYP1-1 T , respectively.Based on the priority of publication, we propose the transfer of Paenihalocynthiibacter styelae (Kim et al., 2021) to the genus Halocynthiibacter as Halocynthiibacter styelae comb.nov.This analysis is consistent with the GTDB taxonomic system (Chaumeil et al., 2019).

Aliiroseovarius
Planktotalea lamellibrachiae DSM 104669 T clearly clustered within the genus Aliiroseovarius (Supplementary Figure S6) and should therefore

Parasedimentitalea and Zongyanglinia
Parasedimentitalea marina W43 T formed a tight cluster with Zongyanglinia huanghaiensis CY05 T and Zongyanglinia marina DSW4-44 T (Figure 3E), sharing AAI values of 82.8 and 82.7%, respectively.The values could justify the classification of the three species into the same genus.This analysis is consistent with the GTDB taxonomic system (Chaumeil et al., 2019).Thus, based on priority, we proposed the transfer of Zongyanglinia huanghaiensis and Zongyanglinia marina to the genus Parasedimentitalea as Parasedimentitalea huanghaiensis comb.nov.and Parasedimentitalea marina comb.nov., respectively.The AAI values of the Parasedimentitalea genomes were 81.8-100%.The genome size of Parasedimentitalea was 4.4-5.6Mb, and the genomic G + C content was 54.2-57.8%.

Meridianimarinicoccus, Phycocomes, and Fluviibacterium
Meridianimarinicoccus roseus TG-679 T , Phycocomes zhengii LMIT002 T , and Fluviibacterium aquatile SM1902 T formed a tight cluster (Figure 3F; Supplementary Figure S5), sharing AAI values of 75.2-98.9%,respectively.We suggest grouping the three species into the same genus.This analysis is consistent with the GTDB taxonomic system (Chaumeil et al., 2019).Thus, based on priority, we propose the transfer of Phycocomes zhengii and Fluviibacterium aquatile to the genus Meridianimarinicoccus as Meridianimarinicoccus zhengii comb.nov.and Meridianimarinicoccus aquatilis comb.nov.The genome size was 3.9-4.6Mbp, and the DNA G + C content was 58.2-67.0%.We also propose the classification of Meridianimarinicoccus in the family Paracoccaceae, instead of the recommended classification in the family Roseobacteraceae (Liang et al., 2021).

Rhodovulum
The 43 Rhodovulum genomes were well grouped (Supplementary Figure S5), sharing AAI values of 72.0-100%.We propose the classification of Rhodovulum as a representative of the family Paracoccaceae, different from the proposed assignment to the family Roseobacteraceae (Liang et al., 2021).

Oceaniglobus and Kandeliimicrobium
Phylogenomic analysis placed Kandeliimicrobium roseum XY-R6 T and Oceaniglobus indicus 1-19b T into the same lineage (Figure 3G).The AAI value between Kandeliimicrobium roseum XY-R6 T and Oceaniglobus indicus 1-19b T was 76.0%.Based on the priority, K. roseum should be re-classified into the genus Oceaniglobus as Oceaniglobus roseus comb.nov.The genome size was 3.7-4.6Mbp, and the genomic G + C content was 59.0-69.0%.We also propose that Oceaniglobus be a representative of the family Paracoccaceae, different from the recommended classification in the family Roseobacteraceae (Liang et al., 2021).

Acidimangrovimonas and Allgaiera
Acidimangrovimonas and Allgaiera were tightly clustered (Supplementary Figure S5).Acidimangrovimonas was first proposed in 2019 with the description of Acidimangrovimonas sediminis (Ren et al., 2019).The authors also re-classified two species of Defluviimonas, Defluviimonas indica, and Defluviimonas pyrenivorans, to the genus Acidimangrovimonas as Acidimangrovimonas indica and Acidimangrovimonas pyrenivorans (Ren et al., 2019).A paper published in 2020 proposed the re-classification of Defluviimonas indica to the genus Allgaiera as Allgaiera indica (Hördt et al., 2020;Oren and Garrity, 2020a).Based on priority, the name Acidimangrovimonas indica has priority and should replace the name Allgaiera indica.The size of the Acidimangrovimonas genomes was 4.3-5.3Mbp with a genomic G + C content of 66.3-67.8%(Supplementary Table S2).The AAI values of the Acidimangrovimonas genomes were 77.5-100%.

Solirhodobacter
This genus currently contains one species with a validly published name, Solirhodobacter olei with type strain Pet-1 T (Chu et al., 2020).The genome size of Solirhodobacter (eight genomes) was 3.1-4.8Mbp with a genomic G + C content of 63.1-69.1%.The AAI value of Solirhodobacter representatives was 72.4-99.8%.

Tabrizicola rongguiensis
Tabrizicola rongguiensis J26 T and an uncultivated bacterium (GCA_945952585.1)formed a clade within the phylogenetic tree that was distinctly separated from other Tabrizicola representatives (Supplementary Figure S5).The AAI value of the two genomes was 89.9% (Supplementary Table S2).Thus, Tabrizicola rongguiensis could be considered a novel genus, and Aliitabrizicola rongguiensis gen.nov., sp.nov. is proposed.The genomic size of Aliitabrizicola is 3.9-4.2Mbp with a genomic G + C content of 64.2-65.2%.

Gemmobacter
The phylogeny Gemmobacter genomes indicated multi-phyletic clades (Figure 3K; Supplementary Figure S5).First, Gemmobacter aquatilis DSM 3857 T and another 15 genomes formed a tight clade, sharing AAI values of 75.7-100%.The ANI and AAI between G. nanjingensis KCTC 23298 T and G. caeni CGMCC 1.7745 T were 98.3 and 99%, respectively, indicating they represented a single species.Based on priority, G. nanjingensis is a later heterotypic synonym of G. caeni.Second, Gemmobacter aestuarii CC-PW-75 T and an uncultivated bacterium ACE_PRO37 (GCA_019454225.1)formed a clade in the phylogenetic tree, which was distinctly separated from other Gemmobacter representatives.The AAI value of Gemmobacter aestuarii CC-PW-75 T and the uncultivated bacterium ACE_PRO37 was 71.6%.Thus, Gemmobacter aestuarii could be considered a representative of a novel genus, for which Aliigemmobacter aestuarii gen.nov., sp.nov. is proposed.The genomic size of Aliigemmobacter is 3.9-4.2Mbp with a DNA G + C content of 64.2-65.2%.Third, Gemmobacter tilapiae KCTC 23310 T formed an independent monophyletic line, which represented a novel genus.Thus, Neogemmobacter gen.nov. is proposed.The type species is Neogemmobacter tilapiae comb.nov.Finally, Rhodobacter ruber

Defluviimonas
The Defluviimonas representatives clustered well in the phylogenetic tree (Supplementary Figure S5).The AAI values among the Defluviimonas representatives were 70.6-100%.

Thioclava
The Thioclava representatives clustered well in the phylogenetic tree (Supplementary Figure S5).The AAI values among the Thioclava representatives were 77.3-100%.

Frigidibacter
The Frigidibacter representatives clustered well in the phylogenetic tree (Supplementary Figure S5).The AAI values among the Frigidibacter representatives were 74.2-100%.

Phaeovulum
The genus Phaeovulum currently contains two species with validly published names, Phaeovulum veldkampii and Phaeovulum vinaykumarii (Parte et 2020;Oren and Garrity, 2020b).However, P. veldkampii DSM 11550 T and P. vinaykumarii JA123 T did not form a cluster in the phylogenetic tree, making us question their placement.
Their taxonomic position needs further study.

Paracoccus
A total of 219 high-quality genomes affiliated to the genus Paracoccus were included in the phylogenomic tree (Figure 3N; Supplementary Figure S5).At the time of writing, the genus contained 81 species with validly published names,8 outnumbering the species of the other genera classified in the family Paracoccaceae.The AAI values among the 219 Paracoccus genomes showed at least six clades, numbered from Clade A to Clade F, to be consistent with the phylogenomic clades (Supplementary Figure S8).This indicated that the genus Paracoccus could be split into several new genera.However, as the genus Paraccocus is widely used, their taxonomic position needs further study.
In summary, based on the above results of phenotypic, genomic, and chemotaxonomic characteristics, strains FR2A1 T and MT2-5-38 represent a novel genus and novel species within the family Paracoccaceae.The name Ostreiculturibacter nitratireducens gen.nov., sp.nov. is proposed, with type strain FR2A1 T (=MCCC 1K08809 T = KCTC 8317 T ).A second strain is MT2-5-38 (=MCCC 1 K08810).Both were isolated from the surface sediment of an oyster farm on a tidal flat in Quanzhou Bay, China.Additionally, based on the AAI values of the above phylogenomic clades, though not all clades in the family Paracoccaceae, the genera have a clear threshold of an AAI value of 70%.Thus, an AAI value of 70% could be considered the genus boundary within the family Paracoccaceae.

Taxonomic consequences
Description of Ostreiculturibacter gen.nov.
Colonies on MB agar plates cultured for 2 days at 30°C are light-white colored, small, and round.Cells are Gram-stainnegative and rod-shaped.Catalase-positive and oxidase-positive.The quinone system is quinone Q-10.The major fatty acids are summed feature 8 (C 18:1 ω7c/C 18:1 ω6c) and C 18:0 .The polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, and several unidentified phospholipids.The genome contained a gene cluster (Sox system) for sulfur oxidation.The genome size is 3.9-4.2Mbp, calculated from the strains and a metagenomeassembled genome.The genomic G + C content is 65.6-69.2%.It was found in the tidal flat surface sediment and the phycosphere of a toxic marine dinoflagellate.The type species is Ostreiculturibacter nitratireducens.The description is as given for the genus, with the following additions.Growth occurred in the temperature range of 20-45°C, with an optimum of 35-40°C.Nitrate can be reduced to nitrite.Positive for tryptophan deaminase.Weak positive for 4-nitrophenylβ-d-galactopyranoside.Positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, and β-glucuronidase; weak positive for lipase (C14), naphthol-AS-BI-phosphohydrolase, and α-glucosidase.The hydrolysis of gelatin and aesculin is negative.Cannot use d-glucose, l-arabinose, d-mannose, d-mannitol, N-acetylglucosamine, d-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetate as sole carbon sources for growth.The genome size is 3.9-4.0Mbp.The genomic G + C content was 65.6-65.7%.The 16S rRNA gene sequence and the whole genome sequence of strain FR2A1 T have been deposited at DDBJ/ENA/GenBank under the accession numbers OR533672 and JAVQHL000000000, respectively.
The type strain is FR2A1 T (=MCCC 1K08809 T = KCTC 8317 T ), and another strain is MT2-5-38 (=MCCC 1 K08810), which were isolated from the surface sediment of an oyster farm on a tidal flat in Quanzhou Bay, China.

Emended description of Pontivivens Park et al. 2015
As the original authors of the genus failed to indicate the gender of the name, and the gender cannot be deduced from the names of its species published thus far, we propose the following emended etymology of the name: Pon.ti.vi'vens.L. masc.n. pontus, the sea; L. pres.part.vivens, living; N.L. neut.n.Pontivivens, an organism living in the sea.
Description of Paramylibacter gen.nov.
Cells are Gram-stain-negative, strictly aerobic, rod-shaped, and non-motile.Catalase-positive and oxidase-positive.The major respiratory quinone is Q-10.The major fatty acid is C 18:1 ω7c.The major polar lipids included phosphatidylglycerol, phosphatidylcholine, and an unidentified aminolipid.Paramylibacter was phylogenetically distinct from Amylibacter marinus.The genomic G + C content was ~49%.The type species is Paramylibacter ulvae.
Paramylibacter kogurei (ko.gu're.i.N.L. gen.n. kogurei, of Kogure, to honor the Japanese microbiologist, Kazuhiro Kogure, in recognition of his contribution to the field of marine microbiology).
Halocynthiibacter styelae (sty.e'lae.N.L. gen.fem.n. styelae, of Styela, named after the generic name of the stalked sea squirt Styela clava, from which the type strain was isolated).
Description of Falsiroseicyclus gen.nov.
Parasedimentitalea huanghaiensis (huang.hai.en'sis.N.L. fem.adj.huanghaiensis, pertaining to Huanghai, the Chinese name for the Yellow Sea, the geographical origin of the type strain).
Description of Parasedimentitalea marina comb.nov.
The description is as given for Kandeliimicrobium roseum (Wang et al., 2018).
Description of Wagnerdoeblera faecalis comb.nov.
Description of Aliitabrizicola gen.nov.The description is as given for Tabrizicola rongguiensis (Xu et al., 2022).
Aliigemmobacter aestuarii (aes.tu.a'ri.i.L. gen.n. aestuarii, of the shallow coast, from where the type strain was isolated).
Description of Neogemmobacter gen.nov.Cells are Gram-stain-negative, aerobic, non-motile, and rod-shaped.Catalase-positive and oxidase-positive.Cells can produce poly-βhydroxybutyrate.The major quinone is Q-10.The major fatty acids are C 18:1 ω7c.The major polar lipids included phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, and unidentified amino lipids.Neogemmobacter formed a separate phylogenetic line with Gemmobacter.The type species is Neogemmobacter tilapiae.

Description of Neogemmobacter tilapiae comb. nov.
Neogemmobacter tilapiae (ti.la'pi.ae.L. gen.n. tilapiae, of Tilapia, the common name of tilapiine cichlid fish, referring to the isolation of the type strain from a pond for rearing Tilapia fish).
Description of Paragemmobacter gen.nov.
Description of Paragemmobacter ruber comb.nov.
Description of Paragemmobacter aquarius comb.nov.
Description of Paenirhodobacter populi comb.nov.

FIGURE 2
FIGURE 2 Phylogenomic tree of the family Paracoccaceae constructed based on bacterial 120 conserved proteins.The bootstrap values on the nodes are displayed by >70.Bar 0.1 represents the amino acid substitution per position.Based on the current taxonomic system in LPSN, Paracoccaceae and Roseobacteraceae are marked as light pink and light green, respectively.The numbers in the brackets are the genomes in the clade.

TABLE 2
Comparison of the cellular fatty acid composition.
Wagnerdoeblera intermedia comb.nov.intermedia(in.ter.me'di.a.L. fem.adj.intermedia, in the middle, referring to the fact that the species is grouped between Gemmobacter and Rhodobacter and Roseinatronobacter and Roseibaca on the basis of 16S rRNA gene sequence similarities).